Mun Hui Sin
Nuraini Binti Abdullah
Siti Rabaah Binti Hamzah
Tan Siew Yen
Method 1 - Callus Culture
Callus is explants that cultured on the appropriate medium,
with auxin and a cytokinin, give rise to an unorganised,
growing and dividing mass of cells.
Any plant tissue that have dividing cells can be used as an
explant.
Exercised plant material is placed on solidified medium
containing nutrients and hormones that promote rapid cell
differentiation.
Callus forms can be quite large (>1cm) (Shuler and Kargi,
2002).
Method 1 - Callus culture
During callus formation there is some degree of
dedifferentiation or changes in morphology and metabolism
that can cause plant cultures to lose the ability to
photosynthesise.
Overcome by the addition of other components such as
vitamins and carbon source to the culture medium.
Method 1 - Callus culture
Figure 1: Function of Auxin and Cytokinin
Auxin Promote root initiation.
Cytokinin Induce growth of shoots
Method 2 - Suspension Culture
There are two categories of callus cultures: compact or
friable. In compact callus, the cells are densely aggregated
while in friable callus the cells are only loosely associated
with each other and the callus becomes soft and breaks apart
easily.
Friable callus provides the inoculum to form cell-suspension
cultures.
The friability of callus is improved by manipulating the
medium components, repeated subculturing or culturing it on
medium with a low concentration of gelling agent.
Method 2 - Suspension Culture
Callus culture is then transferred into liquid medium to
establish suspension culture, which is place on shaker to
supply the cells with sufficient oxygen.
With moderate agitation, cells or small aggregates of cells
will slough off.
A platform shaker is used to give a circular motion in a
viable speed control (30-150 rpm) (Shuler and Kargi, 2002).
Callus culture is performed in the dark (27C & pH 5.5) as
light can encourage differentiation of the callus (Shuler and
Kargi, 2002).
Method 2 - Suspension Culture
During long-term culture, habituation occurs. The culture may
lose the requirement for auxin and/or cytokinin.
Under the correct conditions, these released cells continue to
grow and divide, eventually producing a cell-suspension culture.
The inoculum should not be too large as toxic products released
from damaged or stressed cells can build up to lethal levels.
Cell suspensions can be maintained by batch cultures in conical
flasks. They are continually cultured by repeated subculturing
into fresh medium.
Method 2 - Suspension Culture
This results in dilution of the suspension and the initiation of
another batch growth cycle. The degree of dilution during
subculture should be determined empirically for each culture.
Too great a degree of dilution will result in a greatly extended
lag period or, in extreme cases, death of the transferred cells.
After subculture, the cells divide and the biomass of the culture
increases, until nutrients in the medium are exhausted and/or
toxic by-products build up to inhibitory (stationary phase).
If cells are left in the stationary phase for too long (~2-3 weeks),
they will die and the culture will be lost. Therefore, cells should
be transferred to fresh medium as they enter the stationary phase
(Shuler and Kargi, 2002).
Figure 2: Steps in Plant Cell Culture (Dodds & Roberts, 1995).
Figure 3: Steps in Plant Cell Culture (Shuler and Kargi, 2002).
Figure 4: Callus Culture (right) and Suspension Culture (left) of
Coleus Blumei (Dodds & Roberts, 1995).
References
Dodds, J. H. and Roberts, L. W. (1995). Experiments in plant tissue
culture. Cambridge University Press, Cambridge.
Shuler, M. L. and Kargi, F. (2002). Bioprocess Engineering: Basic
Concept. 2nd Ed. Upper Saddle River, NJ: Prentice Hall PTR
